Human Immunoglobulin G4 (IgG4) ELISpot Antibody Pair Kit is intended for those who want the flexibility of setting up their own ELISpot assay. This kit enables detection and enumeration of cells secreting human IgG4 in a peripheral blood mononuclear cell (PBMC) suspension. ELISpot is commonly used to evaluate immune responses, such as the frequency and isotype or subclass of circulating antigen-specific memory B cells or antibody-secreting cells (ASCs). Soluble IgG4 has been associated with repeated or long-term exposure to protein antigens or allergens. ELISpot is highly sensitive and can detect secreted analytes at a single-cell level. This assay is used in research of infectious diseases and vaccines.
This assay quantifies antigen-specific and/or total IgG4-secreting cells using a modified version of the sandwich ELISA method. Cells are added to ELISpot plates pre-coated with either an antigen of choice or capture antibodies specific for human IgG. Secreted IgG4 is then captured throughout the culture period, before the cells are washed away. Captured IgG4 is detected by the addition of a subclass-specific biotinylated detection antibody, followed by SA-ALP, which binds to the biotinylated antibody. Addition of the chromogenic enzyme substrate 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP)/nitroblue tetrazolium (NBT) results in the formation of colored spots.The number, size, and intensity of these spots are directly proportional to the frequency of antigen-specific or total IgG4-secreting cells in the sample and amount of analyte secreted.
NOTE: This kit includes sufficient reagents for 4 x 96-well ELISpot plates. ELISpot plates (e.g. Millipore Sigma Catalog #MSIPS4W10 / MAIPSWU10D) and BCIP/NBT Substrate (Catalog #200-1016) are required for use with this kit and are available for purchase separately. For detecting antigen-specific immune responses, an antigen of choice is also required.
This kit is supplied in two boxes (Catalog #200-1023/200-1024).
Figure 1. Human IgG4 ELISpot Antibody Pair Kit Detects Total IgG4+ Antibody-Secreting Cells.
ELISpot analysis of peripheral blood mononuclear cells pre-activated with R848, Activator + Recombinant human IL-2, lyophilized (included in kit) or in the absence of stimuli (negative control) at 37°C, 5% CO2 for 4 days. Cells were then seeded at 2 x 10^5 cells/well (R848 + IL-2) or 1 x 10^5 cells/well (no stimuli) on ELISpot plates pre-coated with Anti-human IgG mAb (MT145), unconjugated and incubated again overnight. Following incubation, ELISpot plates were processed as per the instructions on the Product Information Sheet for detecting total IgG4. Wells were imaged and counted using an ELISpot reader.
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